Alkalinization in the isolated and perfused anterior midgut of the larval mosquito, Aedes aegypti

The video (3:35 minutes at four times accelerated speed) shows a typical experiment with an isolated and perfused anterior midgut of larval A. aegypti. Luminal solution: low buffer mosquito saline with m-cresol purple (0.04%). Hemolymph-side solution: high buffer mosquito saline with serotonin (0.2 μmol l−1). The posterior part of the preparation is tied with a human hair onto a perfusion pipette (left) and a glass rod inserted into the lumen (right) maintains the tissue in the focus of the microscope. Luminal perfusion was stopped at the beginning of the video. The color change of m-cresol purple that reflects alkalinization (from yellow to purple) begins at the anterior end of the midgut (right) and continues towards more posterior regions of the anterior midgut (left). The peristaltic muscular activity can also be easily observed (cf. Onken et al. 2004b). At 2:40 minutes, the perfusion pump is started again and the purple, alkaline solution is flushed from the lumen of the preparation.In the present study, isolated midguts of larval Aedes aegypti L. (Diptera: Culicidae) were mounted on perfusion pipettes and bathed in high buffer mosquito saline. With low buffer perfusion saline, containing m-cresol purple, transepithelial voltage was monitored and luminal alkalinization became visible through color changes of m-cresol purple after perfusion stop. Lumen negative voltage and alkalinization depended on metabolic energy and were stimulated in the presence of serotonin (0.2 μmol l−1). In some experiments a pH microelectrode in the lumen recorded pH values up to 10 within minutes after perfusion stop. The V-ATPase inhibitor concanamycin (50 μmol l−1) on the hemolymph side almost abolished Vte and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 μmol l−1), on either the luminal or hemolymph-side, or the inhibitor of anion transport, DIDS (1 mmol l−1) on the luminal side, had no effect on Vte or alkalinization. Cl− substitution in the lumen or on both sides of the tissue affected Vte, but the color change of m-cresol purple was unchanged from control conditions. Hemolymph-side Na+ substitution or addition of the Na+/H+ exchange inhibitor, amiloride (200 μmol l−1), reduced Vte and luminal alkalinization. Luminal amiloride (200 μmol l−1) was without effects on Vte or alkalinization. High K+ (60 mmol l−1) in the lumen reduced Vte without affecting alkalinization. These results indicate that strong luminal alkalinization in isolated and perfused anterior midgut of larval A. aegypti depends on basolateral V-ATPase, but is apparently independent of carbonic anhydrase, apical Cl−/HCO3− exchange or apical K+/2H+ antiport.Financial support for this work was provided by the National Science Foundation (IBN 0091208 to D.F.Moffett and S.B. Moffett) and by the National Institutes of Health (1R01AI063463-01A2 to D.F. Moffett, S.B. Moffett and H. Onken)